1. Field
The invention relates to the genetic differentiation of individual plant isolates and varieties using restriction enzyme technology.
2. State of the Art
A long-standing problem which has plagued plant breeders is the inability to distinguish closely related isolates with any certainty. Both individual and varietal identification are important for a number of purposes including plant breeding and hybridization and patent protection. Effective problem solving in these areas has been severely complicated as it can rarely be demonstrated that one isolate is not just closely related to another, as opposed to being a derivative of it or even an identical isolate.
Presently, differentiation of individual plants or plant varieties is usually accomplished by visual inspection. The two major disadvantages of visual inspection are (a) often there are not enough definitive visual characteristics between closely-related individual plants to make a positive differentiation, and (b) the visual characteristics are greatly affected by environmental factors.
Typical characteristics used in establishing distinctiveness have included habit, immunity from disease, soil condition, color, flavor, productivity, storage qualities, perfume and form. The International Union for the Protection of New Varieties of Plants (UPOV) recommends that competent authorities use the characteristics of color of tuber skin, color of the base of the sprout, color of the tip of the sprout, existence of flowers, color of the inner and outer sides of petals, tuber shape and contour, sprout shape, and stem coloration for the grouping of plant varieties.
Obviously, non-related individual plants may be so visually different that differentiation based on such characteristics is easily accomplished. However, closely-related, yet different, varieties as well as similar progeny in a breeding program will possess many similar structural characteristics which make their distinction by these characteristics nearly impossible. Furthermore, because of the many heritable genes that go together to actually compose a plant's visual appearance and economic characteristics, differences in any one particular gene are often so diffused by the total mass of characters that it cannot be isolated for use as a differentiating characteristic for varietal distinguishment.
Even when sufficient visual characteristics are available to determine individual and varietal differences, they are often unreliable as they are subject to environmental effects. A heritable visual characteristic is usually the result of a phenotypic expression of different plant genes. Often the phenotypic expression of a gene is substantially influenced by environmental factors such as temperature, water condition and amount, soils, amount and intensity of light, available nutrients, etc. This large variation in phenotypic expression may cause severe confusion when the plants are grown under different conditions, as between the distinguishment of plant varieties and the mere recognition of the range of phenotypic expression within a single variety.
Alternatives to visual characteristics for individual and varietal distinguishment have been proposed. One such approach is based on differentiating chemical phenotypic expression through isozyme analyisis. Isozymes are enzymes which have similar biological activities, but have different amino acid sequences. The differences in amino acid sequence indicate that the isozymes originate from different genotypes. Isozyme analysis typically involves the steps of isolation and partial purification of the isozymes of interest with detection of amino acid sequence differences by such techniques as starch gel electrophoresis.
If different isozymes are detected in two different plant isolates, it can always be concluded that they are not genotypically identical. A summation of the similarities and differences between two such isolates can even serve to indicate their overall relatedness. The converse, unfortunately, cannot be concluded. Different plant varieties may not necessarily possess different isozymes. Therefore, one of the major problems with the application of isozyme technology to identification is again the lack of sufficient numbers of variants in closely-related lines to positively distinguish them. In addition, since the isozymes are phenotypic expressions of genes, they are still affected by environmental factors or might only be specific to certain plant tissues.
To avoid the effects of environmental factors and also to have positive identification, one could concentrate on genotypic rather than phenotypic differences. A summation of detectable differences between individual plants in their deoxyribonucleic acid (DNA) sequences could be used as "fingerprints" for estimating the relatedness of those individual plants. Although the technology presently exists to actually sequence DNA, it is not applicable to this problem for practical reasons as it requires a great deal of time and effort to sequence even one short segment of DNA, and this type of analysis would require sequencing many segments from each of many individuals.
Instead of actual DNA sequencing, the present applicants apply restriction fragment technology to detect genomic DNA sequence differences to define relatedness. Although restriction fragment technology and the polymorphisms thus identified have been used to construct genetic linkage maps in humans and other species, it has not been applied to problems in plant science nor developed to detect either varietal differences or homozygosity. The adaptation and application of restriction fragment technology to detect both the degree of relatedness and homozygosity in plants is the essence of the present invention.